Detection of Vancomycin-Resistant Enterococcus spp.

ABSTRACT

The invention provides methods to detect vancomycin-resistant enterococci in biological samples using real-time PCR. Primers and probes for the detection of vancomycin-resistant enterococci are provided by the invention. Articles of manufacture containing such primers and probes for detecting vancomycin-resistant enterococci are further provided by the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of and claims the benefit of priorityunder 35 U.S.C. §120 to U.S. application Ser. No. 10/254,260 having afiling date of Sep. 25, 2002. The disclosure of the prior application isincorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates to bacterial diagnostics, and more particularlyto detection of vancomycin-resistant Enterococcus spp.

BACKGROUND

Enterococci are Gram-positive cocci that are considered normalinhabitants of the gastrointestinal tract and the female genital tract.Enterococcus spp. are not particularly pathogenic in humans, butvancomycin-resistant enterococci have been increasingly identified as animportant cause of hospital acquired infection. Vancomycin-resistantenterococci have been recognized as the second most common cause ofhospital infection, exceeded only by E. coli. Enterococcus faecalis(85-90%) and E. faecium (5-10%) are the species of Enterococci mostcommonly isolated from the gastrointestinal tracts of humans andrepresent the majority of the vancomycin-resistant enterococci.

SUMMARY

The invention provides for methods of identifying vancomycin-resistantenterococci in a biological sample. Primers and probes for detectingnucleic acids encoding vanA, vanB, or vanB-2/3 are provided by theinvention, as are kits containing such primers and probes. Methods ofthe invention can be used to rapidly identify nucleic acids fromvancomycin-resistant enterococci from samples. Using specific primersand probes, the methods include amplifying and monitoring thedevelopment of specific amplification products using real-time PCR.

In one aspect of the invention, there is provided a method for detectingthe presence or absence of one or more vancomycin-resistant nucleicacids in a biological sample from an individual. The method to detectvancomycin-resistant enterococci includes performing at least onecycling step, which includes an amplifying step and a hybridizing step.The amplifying step includes contacting the sample with a pair of vanAprimers to produce an amplification product if a vanA nucleic acidmolecule is present in the sample. The hybridizing step includescontacting the sample with a pair of vanA probes. Generally, the membersof the pair of vanA probes hybridize to the amplification product withinno more than five nucleotides of each other. A first vanA probe of thepair of vanA probes is typically labeled with a donor fluorescent moietyand a second vanA probe of the pair of vanA probes is typically labeledwith a corresponding acceptor fluorescent moiety. The method furtherincludes detecting the presence or absence of fluorescence resonanceenergy transfer (FRET) between the donor fluorescent moiety of the firstvanA probe and the acceptor fluorescent moiety of the second vanA probe.The presence of FRET is usually indicative of the presence of one ormore vancomycin-resistant enterococci in the biological sample, whilethe absence of FRET is usually indicative of the absence of thevancomycin-resistant enterococci in the biological sample.

Alternatively or additionally, the amplifying step can includecontacting the sample with a pair of vanB primers to produce a vanBamplification product if a vanB nucleic acid molecule is present in thesample. The hybridizing step includes contacting the sample with a pairof vanB probes. Generally, the members of the pair of vanB probeshybridize to the amplification product within no more than fivenucleotides of each other. A first vanB probe of the pair of vanB probesis typically labeled with a donor fluorescent moiety and a second vanBprobe of the pair of vanB probes is typically labeled with acorresponding acceptor fluorescent moiety. The method further includesdetecting the presence or absence of FRET between the donor fluorescentmoiety of the first vanB probe and the acceptor fluorescent moiety ofthe second vanB probe. The presence of FRET usually indicates thepresence of one or more vancomycin-resistant enterococci in thebiological sample, and the absence of FRET usually indicates the absenceof a vancomycin-resistant enterococci in the biological sample.

A pair of vanA primers generally includes a first vanA primer and asecond vanA primer The first vanA primer can include the sequence 5′-CGAGGA CGG ATA CAG GA-3′ (SEQ ID NO:1), and the second vanA primer caninclude the sequence 5′-CTT ATC ACC CCT TTA ACG C-3′ (SEQ ID NO:2). Afirst vanA probe can include the sequence 5 ′-CAA GAT AAC GGC CGC ATTGTA CTG AAC GA-3′ (SEQ ID NO:3), and the second vanA probe can includethe sequence 5′-GTC AAT ACT CTG CCC GGT TTC AC-3′ (SEQ ID NO:4).

A pair of vanB/vanB-2/3 primers generally includes a first vanB/vanB-2/3primer and a second vanB/vanB-2/3 primer. A first vanB vanB-2/3 primercan include the sequence 5′-GAA GAT ACC GTG GCT CA-3′ (SEQ ID NO:5), andthe second vanB/vanB-2/3 primer can include the sequence 5′-GTA CGG AAGAAC TTA ACG CT-3′ (SEQ ID NO:6). A first vanB/vanB-2/3 probe can includethe sequence 5′-GAT CCA CTT CGC CGA CAA-3′ (SEQ ID NO:7), and the secondvanB/vanB-2/3 probe can include the sequence 5′-AAA TCA TCC TCG TTT CCCAT-3′ (SEQ ID NO:8).

In some aspects, one of the vanA or vanB/vanB-2/3 primers can be labeledwith a fluorescent moiety (either a donor or acceptor, as appropriate)and can take the place of one of the vanA or vanB/vanB-2/3 probes,respectively.

The members of the pair of vanA probes or vanB/vanB-2/3 probes canhybridize within no more than two nucleotides of each other, or canhybridize within no more than one nucleotide of each other. Arepresentative donor fluorescent moiety is fluorescein, andcorresponding acceptor fluorescent moieties include LC-Red 640, LC-Red705, Cy5, and Cy5.5. Additional corresponding donor and acceptorfluorescent moieties are known in the art.

In one aspect, the detecting step includes exciting the biologicalsample at a wavelength absorbed by the donor fluorescent moiety andvisualizing and/or measuring the wavelength emitted by the acceptorfluorescent moiety (i.e., visualizing and/or measuring FRET). In anotheraspect, the detecting comprises quantitating the FRET. In yet anotheraspect, the detecting step can be performed after each cycling step(i.e., in real-time).

Generally, the presence of FRET within 50 cycles (e.g., 20, 25, 30, 35,40, or 45 cycles) indicates the presence of one or morevancomycin-resistant enterococci in the individual. In addition,determining the melting temperature between one or both of the vanAprobe(s) and the amplification product, wherein the melting temperatureconfirms the presence or the absence of a vancomycin-resistantenterococci, while determining the melting temperature between one orboth of the vanB/vanB-2/3 probe(s) and the vanB/vanB-2/3 amplificationproduct also confirms the presence or the absence of particularvancomycin-resistant enterococci.

Representative biological samples include anal or perirectal swabs,stool samples, blood, and body fluids. The above-described methods canfurther include preventing amplification of a contaminant nucleic acid.Preventing amplification of a contaminant nucleic acid can includeperforming the amplifying step in the presence of uracil and treatingthe biological sample with uracil-DNA glycosylase prior to amplifying.

In addition, the cycling step can be performed on a control sample. Acontrol sample can include a vanA, vanB, or vanB-2/3 nucleic acidmolecule. Alternatively, a control sample can include a nucleic acidmolecule other than a vanA, vanB or vanB-2/3 nucleic acid molecule.Cycling steps can be performed on such a control sample using a pair ofcontrol primers and a pair of control probes. The control primers andthe control probes are other than the vanA or vanB/vanB-2/3 primers andprobes. One or more amplifying steps can produce a control amplificationproduct. Each of the control probes hybridizes to the controlamplification product.

In another aspect of the invention, there are provided articles ofmanufacture, or kits. Kits of the invention can include a pair of vanAprimers; a pair of vanA probes; and a donor fluorescent moiety and acorresponding fluorescent moiety. For example, a first vanA primerprovided in a kit of the invention can include the sequence 5′-CGA GGACGG ATA CAG GA-3′ (SEQ ID NO:1), and the second vanA primer can includethe sequence 5′-CTT ATC ACC CCT TTA ACG C-3′ (SEQ ID NO:2). A first vanAprobe can include the sequence 5′-CAA GAT AAC GGC CGC ATT GTA CTG AACGA-3′ (SEQ ID NO:3), and the second vanA probe can include the sequence5′-GTC AAT ACT CTG CCC GGT TTC AC-3′ (SEQ I) NO:4).

Articles of manufacture of the invention can further or alternativelyinclude a pair of vanB/vanB-2/3 primers; a pair of vanB/vanB-2/3 probes;and a donor fluorescent moiety and a corresponding fluorescent moiety.For example, the first vanB/vanB-2/3 primer provided in a kit of theinvention can include the sequence 5′-GAA GAT ACC GTG GCT CA-3′ (SEQ IDNO:5), and the second vanB/vanB-2/3 primer can include the sequence5′-GTA CGG AAG AAC TTA ACG CT-3′ (SEQ ID NO:6). The first vanB/vanB-2/3probe provided in a kit of the invention can include the sequence 5′-GATCCA CTT CGC CGA CAA-3′ (SEQ ID NO:7), and the second vanB/vanB-2/3 probecan include the sequence 5′-AAA TCA TCC TCG TTT CCC AT-3′ (SEQ ID NO:8).

Articles of the invention can include fluorophoric moieties for labelingthe probes, or probes already labeled with donor and correspondingacceptor fluorescent moieties. The article of manufacture can alsoinclude a package label or package insert having instructions thereonfor using the pair of vanA primers and the pair of vanA probes to detectthe presence or absence of one or more vancomycin-resistant enterococciin a biological sample, or for using the pair of vanB/vanB-2/3 primersand the pair of vanB/vanB-2/3 probes to detect the presence or absenceof one or more vancomycin-resistant enterococci in a biological sample.

In yet another aspect of the invention, there is provided a method fordetecting the presence or absence of one or more vancomycin-resistantenterococci in a biological sample from an individual. Such a methodincludes performing at least one cycling step. A cycling step caninclude an amplifying step and a hybridizing step. Generally, anamplifying step includes contacting the sample with a pair of vanAprimers to produce an amplification product if a vanA nucleic acidmolecule is present in the sample. Generally, a hybridizing stepincludes contacting the sample with a vanA probe. Such a vanA probe isusually labeled with a donor fluorescent moiety and a correspondingacceptor fluorescent moiety. The method further includes detecting thepresence or absence of FRET between the donor fluorescent moiety and theacceptor fluorescent moiety of the vanA probe. The presence or absenceof FRET is indicative of the presence or absence of one or morevancomycin-resistant enterococci in the sample.

In one aspect, amplification can employ a polymerase enzyme having 5′ to3′ exonuclease activity. Thus, the donor and acceptor fluorescentmoieties would be within no more than 5 nucleotides of each other alongthe length of the probe. In another aspect, the vanA probe includes anucleic acid sequence that permits secondary structure formation. Suchsecondary structure formation generally results in spatial proximitybetween the donor and the acceptor fluorescent moiety. According to thismethod, the acceptor fluorescent moiety is a quencher.

In another aspect of the invention, there is provided a method fordetecting the presence or absence of one or more vancomycin-resistantenterococci in a biological sample from an individual. Such a methodincludes performing at least one cycling step. A cycling step caninclude an amplifying step and a dye-binding step. An amplifying stepgenerally includes contacting the sample with a pair of vanA primers toproduce an amplification product if a vanA nucleic acid molecule ispresent in the sample. A dye-binding step generally includes contactingthe amplification product with a double-stranded nucleic acid bindingdye. The method further includes detecting the presence or absence ofbinding of the double-stranded nucleic acid binding dye to theamplification product. According to the invention, the presence ofbinding is typically indicative of the presence of one or morevancomycin-resistant enterococci in the sample, and the absence ofbinding is typically indicative of the absence of a vancomycin-resistantenterococci in the sample. Such a method can further include the stepsof determining the melting temperature between the amplification productand the double-stranded nucleic acid binding dye. Generally, the meltingtemperature confirms the presence or absence of vancomycin-resistantenterococci. Representative double-stranded nucleic acid binding dyeinclude SYBRGreenI®, SYBRGold®, and ethidium bromide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of vanA nucleic acid sequences.

FIG. 2 is an alignment of vanB nucleic acid sequences.

DETAILED DESCRIPTION

A real-time PCR assay that is more sensitive than existing assays isdescribed herein for detecting vancomycin-resistant enterococci in abiological sample such as a fecal sample. Primers and probes fordetecting vanA, vanB, and/or vanB-2/3 nucleic acids are provided by theinvention, as are articles of manufacture containing such primers andprobes. The assay for vancomycin-resistant enterococci was designed todiscriminate between the vanA, vanB and vanB-2/3 genotypes based on adifference in melting temperature between a pair of vanA probes and apair of vanB probes. The increased sensitivity of the real-time PCRassay for detecting vancomycin-resistant enterococci nucleic acidscompared to other methods, as well as the improved features of real-timePCR including sample containment and real-time detection of theamplified product, make feasible the implementation of this technologyfor routine clinical laboratory for the detection ofvancomycin-resistant enterococci.

The total time for processing a sample using the LIGHTCYCLER™vancomycin-resistant Enteroccocus assay is less than 3 hrs compared to4-7 days for detection by routine culture. The invention has thepotential to replace standard culture methods which require selectmedia, biochemical testing, and susceptibility testing and therefore,result in cost savings to institutions. Since clinicians receive asingle test result within a few hours, appropriate isolation proceduresand antimicrobial therapy can begin almost immediately. The rapidvancomycin-resistant Enteroccocus real-time PCR assay allows hospitalsto take the necessary precautions with vancomycin-resistantenterococci-infected patients such that the spread ofvancomycin-resistant enterococci to other patients is prevented.

Enterococcus spp.

Enterococcus spp. have the characteristic of being resistant to manyantimicrobial agents, which make them formidable pathogens and limit thetherapeutic options available to the clinician. All enterococci areintrinsically resistant to a number of antibiotics and exhibit lowlevels of resistance to the β-lactam agents, the aminogycosides, and thelincosamides. They have acquired genes of resistance to all knownantimicrobial agents, including the glycopeptides vancomycin andteicoplanin. One of the concerns is the possibility that thevancomycin-resistant genes may be transferred to other Gram-positiveorganisms, especially Staphylococcus aureus.

A BLAST alignment of vanA sequences found no other organisms containingsequences similar to vanA. A BLAST alignment of vanB sequences showedthat vanB sequences can be found in Enterococcus spp. and animal species(veal calves) of streptococci such as S. gallolyticus and S. infantarius(Genebank Accession Nos. AY035705 and Z70527). One other isolate, S.bovis, also has sequences that exhibit homology to vanB sequences. Thesestreptococcus isolates appear to have acquired enterococcal vanBvancomycin resistance genes.

Vancomycin-resistant enterococci exhibit optimal growth at 35° C. andwill grow in 6.5% NaCl. Vancomycin-resistant enterococci are able tohydrolyze esculin. Vancomycin-resistant enterococci are selectivelycultured on Enterococcosel agar containing 8 μg/ml vancomycin. Theglycopeptide resistance of vancomycin-resistant enterococci has threedifferent phenotypes. vanA is the most frequently isolated phenotypewith high levels of resistance to vancomycin and teicoplanin. The vanBphenotypes (e.g., vanB, or vanB-2/3) has variable vancomycin-resistanceand is susceptible to teicoplanin. The vanC phenotype has low levels ofvancomycin-resistance and is susceptible to teicoplanin and is thereforeless important for detection by a clinical laboratory.

PCR-RFLP assays following Mspl restriction digestion can be used todifferentiate the vanA genotype from the vanB genotype. The vanA strainstypically exhibit a high level of vancomycin resistance (minimuminhibitory concentration (MIC)>64 μg/ml). vanA strains also exhibitinducible resistance to vancomycin and teicoplanin. The genes encodingvanA are located on a transposon or a plasmid, and are easilytransferred by conjugation. The first vanA strain ofvancomycin-resistant enterococci was reported in 1986, and representsapproximately 70% of vancomycin-resistant enterococci isolates frompatient specimens. On the other hand, vanB strains exhibit variableresistance to vancomycin (MIC 4 to >1024 μg/ml), and exhibit inducibleresistance to vancomycin only. The genes encoding vanB are chromosomaland can be transferred by conjugation. vanB strains were firstidentified in the U.S. in 1987, and currently make up about 25% of thevancomycin-resistant patient isolates.

Vancomycin-resistant Enterococci Nucleic Acids and Oligonucleotides

The invention provides methods to detect vanA, vanB, and/or van-B2/3nucleic acids by amplifying, for example, nucleic acid moleculescorresponding to a portion of vanA, vanB, and/or vanB-2/3. Nucleic acidmolecules other than those exemplified herein (e.g., other than vanA,vanB, and/or vanB-2/3) also can be used to detect vancomycin-resistantenterococci in a sample and are known to those of skill in the art.vanA, vanB, and vanB-2/3 nucleic acid sequences have been described(see, for example, GenBank Accession Nos. M97297, U94528, and U72704).Specifically, primers and probes to amplify and detect vanA, vanB,and/or vanB-2/3 nucleic acids are provided by the invention.

Primers that amplify a vanA, vanB, and/or vanB-2/3 nucleic acidmolecule, e.g., nucleic acids encoding a portion of vanA, vanB, and/orvanB-2/3, can be designed using, for example, a computer program such asOLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Importantfeatures when designing oligonucleotides to be used as amplificationprimers include, but are not limited to, an appropriate sizeamplification product to facilitate detection (e.g., byelectrophoresis), similar melting temperatures for the members of a pairof primers, and the length of each primer (i.e., the primers need to belong enough to anneal with sequence-specificity and to initiatesynthesis but not so long that fidelity is reduced duringoligonucleotide synthesis). Typically, oligonucleotide primers are 8 to50 nucleotides in length (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).“vanA primers”, “vanB primers”, or “vanB2/3 primers” as used hereinrefer to oligonucleotide primers that anneal to vanA, vanB, and/orvanB2/3 nucleic acid sequences, respectively, and initiate synthesistherefrom under appropriate conditions.

Designing oligonucleotides to be used as hybridization probes can beperformed in a manner similar to the design of primers, although themembers of a pair of probes preferably anneal to an amplificationproduct within no more than 5 nucleotides of each other on the samestrand such that fluorescent resonance energy transfer (FRET) can occur(e.g., within no more than 1, 2, 3, or 4 nucleotides of each other).This minimal degree of separation typically brings the respectivefluorescent moieties into sufficient proximity such that FRET occurs. Itis to be understood, however, that other separation distances (e.g., 6or more nucleotides) are possible provided the fluorescent moieties areappropriately positioned relative to each other (for example, with alinker arm) such that FRET can occur. In addition, probes can bedesigned to hybridize to targets that contain a mutation orpolymorphism, thereby allowing differential detection ofvancomycin-resistant enterococci based on either absolute hybridizationof different pairs of probes corresponding to each particularenterococci to be distinguished or differential melting temperaturesbetween, for example, members of a pair of probes and each amplificationproduct generated from a vancomycin-resistant enterococci. As witholigonucleotide primers, oligonucleotide probes usually have similarmelting temperatures, and the length of each probe must be sufficientfor sequence-specific hybridization to occur but not so long thatfidelity is reduced during synthesis. Oligonucleotide probes typicallyare 8 to 50 nucleotides in length (e.g., 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides inlength). “vanA probes” as used herein refers to oligonucleotide probesthat specifically anneal to vanA amplification products. As used herein,“vanB/vanB-2/3 probes” refer to oligonucleotide probes that anneal toeither vanB or vanB-2/3, which can be differentiated based upon themelting temperature of the vanB/vanB-2/3 probes with the respective(i.e., vanB or vanB-2/3) amplification product.

Constructs of the invention include vectors containing a vanA, vanB,and/or vanB-2/3 nucleic acid molecule, e.g., a vanA, vanB, and/orvanB-2/3 gene, or fragment thereof. Constructs can be used, for example,as control template nucleic acids. Vectors suitable for use in thepresent invention are commercially available or can be produced byrecombinant DNA technology methods routine in the art. A vanA, vanB,and/or vanB-2/3 nucleic acid molecule can be obtained, for example, bychemical synthesis, direct cloning from a vancomycin-resistantenterococci, or by PCR amplification. A vancomycin-resistantEnterococcus nucleic acid molecule or fragments thereof can be operablylinked to a promoter or other regulatory element such as an enhancersequence, a response element or an inducible element that modulatesexpression of the Enterococcus nucleic acid molecule. As used herein,operably linking refers to connecting a promoter and/or other regulatoryelements to an Enterococcus nucleic acid molecule in such a way as topermit and/or regulate expression of the nucleic acid molecule. Apromoter that does not normally direct expression of vanA, vanB, and/orvanB-2/3 can be used to direct transcription of a vanA, vanB, and/orvanB-2/3 nucleic acid molecule using, for example a viral polymerase, abacterial polymerase, or a eukaryotic RNA polymerase. Alternatively, avanA, vanB, and/or vanB-2/3 native promoter can be used to directtranscription of a vanA, vanB, and/or vanB-2/3 nucleic acid moleculeusing, for example, an E. coli RNA polymerase or a host RNA polymerase.In addition, operably linked can refer to an appropriate connectionbetween a vanA, vanB, and/or vanB-2/3 promoter or other regulatoryelement to a heterologous coding sequence (i.e., a non-vanA, -vanB,and/or -vanB-2/3 coding sequence, for example a reporter gene) in such away as to permit expression of the heterologous coding sequence.

Constructs suitable for use in the methods of the invention typicallyinclude, in addition to a vanA, vanB, and/or vanB-2/3 nucleic acidmolecule, sequences encoding a selectable marker (e.g., an antibioticresistance gene) for selecting desired constructs and/or transformants,and an origin of replication. The choice of vector systems usuallydepends upon several factors, including, but not limited to, the choiceof host cells, replication efficiency, selectability, inducibility, andthe ease of recovery.

Constructs of the invention containing a vanA, vanB, and/or vanB-2/3nucleic acid molecule can be propagated in a host cell. As used herein,the term host cell is meant to include prokaryotes and eukaryotes.Prokaryotic hosts can include E. coli, Salmonella typhimurium, Serratiamarcescens and Bacillus subtilis. Eukaryotic hosts include yeasts suchas S. cerevisiae, S. pombe, and Pichia pastoris, mammalian cells such asCOS cells or Chinese hamster ovary (CHO) cells, insect cells, and plantcells such as Arabidopsis thaliana and Nicotiana tabacum. A construct ofthe invention can be introduced into a host cell using any of thetechniques commonly known to those of ordinary skill in the art. Forexample, calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transferare common methods for introducing nucleic acids into host cells. Inaddition, naked DNA can be delivered directly to cells (see, e.g., U.S.Pat. Nos. 5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in the present invention include oligonucleotidescapable of acting as points of initiation of nucleic acid synthesiswithin a vancomycin-resistant enterococci nucleic acid sequence. Aprimer can be purified from a restriction digest by conventionalmethods, or it can be produced synthetically. A primer is preferablysingle-stranded for maximum efficiency in amplification, but a primercan be double-stranded. Double-stranded primers are first denatured,i.e., treated to separate the strands. One method of denaturingdouble-stranded nucleic acids is by heating.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCRprovided the enzyme is replenished.

If the nucleic acid template is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 0 sec to 4 min.

If the double-stranded nucleic acid is denatured by heat, the reactionmixture is allowed to cool to a temperature that promotes annealing ofeach primer to its target sequence on the vancomycin-resistantenterococci nucleic acid. The temperature for annealing is usually fromabout 35° C. to about 65° C. The reaction mixture is then adjusted to atemperature at which the activity of the polymerase is promoted oroptimized, e.g., a temperature sufficient for extension to occur fromthe annealed primer to generate products complementary to the templatenucleic acid. The temperature should be sufficient to synthesize anextension product from each primer that is annealed to a nucleic acidtemplate, but should not be so high as to denature an extension productfrom its complementary template. The temperature generally ranges fromabout 40° to 80° C.

PCR assays can employ nucleic acid template such as DNA or RNA,including messenger RNA (mRNA). The template nucleic acid need not bepurified; it may be a minor fraction of a complex mixture, such asvancomycin-resistant Enterococcus nucleic acid contained in human cells.DNA or RNA may be extracted from any biological sample such as stool,anal or perirectal swabs, or body fluids (e.g., blood or urine) byroutine techniques such as those described in Diagnostic MolecularMicrobiology: Principles and Applications (Persing et al. (eds), 1993,American Society for Microbiology, Washington D.C.). vanA, vanB, and/orvanB-2/3 nucleic acids to be used as controls can be obtained from anynumber of sources, such as plasmids, or natural sources includingbacteria, yeast, viruses, organelles, or higher organisms such as plantsor animals.

The oligonucleotide primers are combined with other PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH8.3), 1.5 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO. The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof. In certaincircumstances, 300 to 640 μM dUTP can be substituted for dTTP in thereaction.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity amplification products corresponding tothe target vancomycin-resistant enterococci nucleic acid molecule. Thelimiting factors in the reaction are the amounts of primers,thermostable enzyme, and nucleoside triphosphates present in thereaction. The cycling steps (i.e., amplification and hybridization) arepreferably repeated at least once. For use in detection, the number ofcycling steps will depend, e.g., on the nature of the sample. If thesample is a complex mixture of nucleic acids, more cycling steps may berequired to amplify the target sequence sufficient for detection.Generally, the cycling steps are repeated at least about 20 times, butmay be repeated as many as 40, 60, or even 100 times.

Fluorescent Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on the fact that when a donor and acorresponding acceptor fluorescent moiety are positioned within acertain distance of each other, energy transfer takes place between thetwo fluorescent moieties that can be visualized or otherwise detectedand/or quantitated. As used herein, two oligonucleotide probes, eachcontaining a fluorescent moiety, can hybridize to an amplificationproduct at particular positions determined by the complementarity of theoligonucleotide probes to the vancomycin-resistant enterococci targetnucleic acid sequence. Upon hybridization of the oligonucleotide probesto the amplification product at the appropriate positions, a FRET signalis generated.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system or afluorometer. Excitation to initiate energy transfer can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptorfluorescent moieties, “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety preferably should be atleast 100 nm greater than the wavelength maximum of the excitationspectrum of the donor fluorescent moiety. Accordingly, efficientnon-radiative energy transfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 um), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC™-Red 640, LC™-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm can be important, as the linker arms will affect the distancebetween the donor and the acceptor fluorescent moieties. The length of alinker arm for the purpose of the present invention is the distance inAngstroms (Å) from the nucleotide base to the fluorescent moiety. Ingeneral, a linker arm is from about 10 to about 25 Å. The linker arm maybe of the kind described in WO 84/03285. WO 84/03285 also disclosesmethods for attaching linker arms to particular nucleotide bases, andalso for attaching fluorescent moieties to a linker arm.

An acceptor fluorescent moiety such as an LC™-Red 640-NHS-ester can becombined with C6-Phosphoramidites (available from ABI (Foster City,Calif.) or Glen Research (Sterling, Va.)) to produce, for example,LC™-Red 640-Phosphoramidite. Frequently used linkers to couple a donorfluorescent moiety such as fluorescein to an oligonucleotide includethiourea linkers (FITC-derived, for example, fluorescein-CPG's from GlenResearch or ChemGene (Ashland, Mass.)), amide-linkers(fluorcscein-NHS-ester-derived, such as fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPG's that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of Vancomycin-resistant Enterococci

In the hospital laboratory, routine culture for the detection ofvancomycin-resistant Enterococcus from stool or anal swabs usingselective media is a reliable method but may require up to 4-7 days foridentification. Culture methods are also time consuming and expensivefor laboratories performing a large number of specimens. For recovery ofvancomycin-resistant enterococci in the laboratory, a selective mediumcontaining vancomycin at a concentration of 8 μg/ml in agar is used.This medium also contains bile esculin, which is hydrolyzed to impart ablack-brown color to Enterococcus colonies. Identification of suspectcolonies and antimicrobial susceptibility tests are performed onEnterococcus spp., which also can take several days to perform.

The invention provides methods for detecting the presence or absence ofone or more vancomycin-resistant enterococci in a biological sample froman individual. Methods provided by the invention avoid problems ofsample contamination, false negatives and false positives. The methodsinclude performing at least one cycling step that includes amplifyingand hybridizing. An amplification step includes contacting thebiological sample with a pair of vanA, vanB, and/or vanB-2/3 primers toproduce an amplification product if nucleic acid molecules from one ormore vancomycin-resistant enterococci are present in the sample. Each ofthe vanA, vanB, and/or vanB-2/3 primers anneals to a target within oradjacent to a vanA, vanB, and/or vanB-2/3 nucleic acid molecule,respectively, such that at least a portion of the amplification productcontains nucleic acid sequence corresponding to the respectivevancomycin-resistant enterococci nucleic acid, and, more importantly,such that the amplification product contains the nucleic acid sequencesthat are complementary to vanA, vanB, and/or vanB-2/3 probes. Ahybridizing step includes contacting the sample with a pair of vanA,vanB, and/or vanB2/3 probes. Generally, the members of the pair of vanA,vanB, and/or vanB-2/3 probes hybridize to the appropriate amplificationproduct within no more than five nucleotides of each other. According tothe invention, a first vanA, vanB, and/or vanB-2/3 probe of the pair ofvanA, vanB, and/or vanB-2/3 probes, respectively, is labeled with adonor fluorescent moiety and a second vanA, vanB, and/or vanB-2/3 probeof the pair of vanA, vanB, and/or vanB-2/3 probes, respectively, islabeled with a corresponding acceptor fluorescent moiety. The methodfurther includes detecting the presence or absence of FRET between thedonor fluorescent moiety of the first vanA, vanB, and/or vanB-2/3 probeand the corresponding acceptor fluorescent moiety of the second vanA,vanB, and/or vanB-2/3 probe. Multiple cycling steps can be performed,preferably in a thermocycler. The above-described methods for detectingvancomycin-resistant Enterococcus in a biological sample using primersand probes directed toward vanA, vanB, and/or vanB-2/3 also can beperformed using other vancomycin-resistant Enterococcus gene-specificprimers and probes.

As used herein, “amplifying” refers to the process of synthesizingnucleic acid molecules that are complementary to one or both strands ofa template nucleic acid (e.g., vanA, vanB, and/or vanB-2/3 nucleic acidmolecules). Amplifying a nucleic acid molecule typically includesdenaturing the template nucleic acid, annealing primers to the templatenucleic acid at a temperature that is below the melting temperatures ofthe primers, and enzymatically elongating from the primers to generatean amplification product. The denaturing, annealing and elongating stepseach can be performed once. Generally, however, the denaturing,annealing and elongating steps are performed multiple times such thatthe amount of amplification product is increasing, often timesexponentially, although exponential amplification is not required by thepresent methods. Amplification typically requires the presence ofdeoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g.,PLATINUM® TAQ) and an appropriate buffer and/or co-factors for optimalactivity of the polymerase enzyme (e.g., MgCl₂ and/or KCl).

If amplification of vancomycin-resistant enterococci nucleic acid occursand an amplification product is produced, the step of hybridizingresults in a detectable signal based upon FRET between the members ofthe pair of probes. As used herein, “hybridizing” refers to theannealing of probes to an amplification product. Hybridizationconditions typically include a temperature that is below the meltingtemperature of the probes but that avoids non-specific hybridization ofthe probes.

Generally, the presence of FRET indicates the presence ofvancomycin-resistant enterococci in the biological sample, and theabsence of FRET indicates the absence of a vancomycin-resistantenterococci in the biological sample. Inadequate specimen collection,transportation delays, inappropriate transportation conditions, or useof certain collection swabs (e.g., calcium alginate or aluminum shaft)are all conditions that can affect the success and/or accuracy of thetest result, however. Using the methods disclosed herein, detection ofFRET within 45 cycling steps is indicative of one or morevancomycin-resistant enterococci.

Representative biological samples that can be used in practicing themethods of the invention include anal or perirectal swabs, stoolsamples, blood, or body fluids. Biological sample collection and storagemethods are known to those of skill in the art. Biological samples canbe processed (e.g., by standard nucleic acid extraction methods and/orusing commercial kits) to release nucleic acid encodingvancomycin-resistance or, in some cases, the biological sample iscontacted directly with the PCR reaction components and the appropriateoligonucleotides.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the vanA, vanB, and/or vanB-2/3 probes fromthe respective amplification product, respectively, can confirm thepresence of one or more vancomycin-resistant enterococci in the sample.

Within each thermocycler run, control samples can be cycled as well.Control nucleic acid template can be amplified from a positive controlsample (e.g., template other than vanA, vanB, and/or vanB-2/3) using,for example, control primers and control probes. Positive controlsamples can also be used to amplify, for example, a plasmid constructcontaining a vancomycin-resistant enterococci nucleic acid molecule.Such a plasmid control can be amplified internally (e.g., within eachbiological sample) or in separate samples run side-by-side with thepatients' samples. Each thermocycler run also should include a negativecontrol that, for example, lacks vancomycin-resistant enterococcitemplate nucleic acid. Such controls are indicators of the success orfailure of the amplification, hybridization, and/or FRET reaction.Therefore, control reactions can readily determine, for example, theability of primers to anneal with sequence-specificity and to initiateelongation, as well as the ability of probes to hybridize withsequence-specificity and for FRET to occur.

In one embodiment, the methods of the invention include steps to avoidcontamination. For example, an enzymatic method utilizing uracil-DNAglycosylase is described in U.S. Pat. Nos. 5,035,996, 5,683,896 and5,945,313 to reduce or eliminate contamination between one thermocyclerrun and the next. In addition, standard laboratory containment practicesand procedures are desirable when performing methods of the invention.Containment practices and procedures include, but are not limited to,separate work areas for different steps of a method, containment hoods,barrier filter pipette tips and dedicated air displacement pipettes.Consistent containment practices and procedures by personnel aredesirable for accuracy in a diagnostic laboratory handling clinicalsamples.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods of the invention. In one embodiment, aLIGHTCYCLER™ instrument is used. A detailed description of theLIGHTCYCLER™ System and real-time and on-line monitoring of PCR can befound at biochem.roche.com/lightcycler on the World Wide Web. Thefollowing patent applications describe real-time PCR as used in theLIGHTCYCLER™ technology: WO 97/46707, WO 97/46714 and WO 97/46712. TheLIGHTCYCLER™ instrument is a rapid thermocycler combined with amicrovolume fluorometer utilizing high quality optics. This rapidthermocycling technique uses thin glass cuvettes as reaction vessels.Heating and cooling of the reaction chamber are controlled byalternating heated and ambient air. Due to the low mass of air and thehigh ratio of surface area to volume of the cuvettes, very rapidtemperature exchange rates can be achieved within the LIGHTCYCLER™thermal chamber. Addition of selected fluorescent dyes to the reactioncomponents allows the PCR to be monitored in real-time and on-line.Furthermore, the cuvettes serve as an optical element for signalcollection (similar to glass fiber optics), concentrating the signal atthe tip of the cuvettes. The effect is efficient illumination andfluorescent monitoring of microvolume samples.

The LIGHTCYCLER™ carousel that houses the cuvettes can be removed fromthe instrument. Therefore, samples can be loaded outside of theinstrument (in a PCR Clean Room, for example). In addition, this featureallows for the sample carousel to be easily cleaned and sterilized. Thefluorometer, as part of the LIGHTCYCLER™ apparatus, houses the lightsource. The emitted light is filtered and focused by an epi-illuminationlens onto the top of the cuvettes. Fluorescent light emitted from thesample is ten focused by the same lens, passed through a dichroicmirror, filtered appropriately, and focused onto data-collectingphotohybrids. The optical unit currently available in the LIGHTCYCLER™instrument (Catalog No. 2 011 468) includes three band-pass filters (530nm, 640 nm, and 710 nm), providing three-color detection and severalfluorescence acquisition options. Data collection options include onceper cycling step monitoring, fully continuous single-sample acquisitionfor melting curve analysis, continuous sampling (in which samplingfrequency is dependent on sample number) and/or stepwise measurement ofall samples after defined temperature interval.

The LIGHTCYCLER™ can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 msec. After each cycling step, a quantitative display offluorescence vs. cycle number can be continually updated for allsamples. The data generated can be stored for further analysis.

A common FRET technology format utilizes two hybridization probes. Eachprobe can be labeled with a different fluorescent moiety and the twoprobes are generally designed to hybridize in close proximity to eachother in a target DNA molecule (e.g., an amplification product). By wayof example, a donor fluorescent moiety such as fluorescein can beexcited at 470 nm by the light source of the LIGHTCYCLER™ Instrument.During FRET, fluorescein transfers its energy to an acceptor fluorescentmoiety such as LIGHTCYCLER™-Red 640 (LC™-Red 640) or LIGHTCYCLER™-Red705 (LC™-Red 705). The acceptor fluorescent moiety then emits light of alonger wavelength (e.g., 640 nm or 705 nm, respectively), which isdetected by the optical detection system of the LIGHTCYCLER™ instrument.Other donor and corresponding acceptor fluorescent moieties suitable foruse in the invention arc described above. Efficient FRET can only takeplace when the fluorescent moieties are in direct local proximity (forexample, within 5 nucleotides of each other as described above) and whenthe emission spectrum of the donor fluorescent moiety overlaps with theabsorption spectrum of the acceptor fluorescent moiety. The intensity ofthe emitted signal can be correlated with the number of original targetDNA molecules (e.g., the number of vancomycin-resistant enterococci).

Another FRET technology format utilizes TAQMAN® technology to detect thepresence or absence of an amplification product, and hence, the presenceor absence of vancomycin-resistant enterococci. TAQMAN® technologyutilizes one single-stranded hybridization probe labeled with twofluorescent moieties. When a first fluorescent moiety is excited withlight of a suitable wavelength, the absorbed energy is transferred to asecond fluorescent moiety according to the principles of FRET. Thesecond fluorescent moiety is generally a quencher molecule. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product) and isdegraded by the 5′ to 3′ exonuclease activity of the Taq polymeraseduring the subsequent elongation phase. As a result, the excitedfluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.) uses TAQMAN® technology, and issuitable for performing the methods described herein for detectingvancomycin-resistant enterococci. Information on PCR amplification anddetection using an ABI PRISM® 770 system can be found atappliedbiosystems.com/products on the World Wide Web.

Yet another FRET technology format utilizes molecular beacon technologyto detect the presence or absence of an amplification product, andhence, the presence or absence of one or more vancomycin-resistantenterococci. Molecular beacon technology uses a hybridization probelabeled with a donor fluorescent moiety and an acceptor fluorescentmoiety. The acceptor fluorescent moiety is generally a quencher, and thefluorescent labels are typically located at each end of the probe.Molecular beacon technology uses a probe oligonucleotide havingsequences that permit secondary structure formation (e.g., a hairpin).As a result of secondary structure formation within the probe, bothfluorescent moieties are in spatial proximity when the probe is insolution. After hybridization to the target nucleic acids (i.e., theamplification products), the secondary structure of the probe isdisrupted and the fluorescent moieties become separated from one anothersuch that after excitation with light of a suitable wavelength, theemission of the first fluorescent moiety can be detected.

As an alternative to detection using FRET technology, an amplificationproduct can be detected using a nucleic acid binding dye such as afluorescent DNA binding dye (e.g., SYBRGreenI® or SYBRGold® (MolecularProbes)). Upon interaction with the double-stranded nucleic acid, suchnucleic acid binding dyes emit a fluorescence signal after excitationwith light at a suitable wavelength. A nucleic acid binding dye such asa nucleic acid intercalating dye also can be used. When nucleic acidbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

It is understood that the present invention is not limited by theconfiguration of one or more commercially available instruments.

Articles of Manufacture

The invention further provides for articles of manufacture to detectvancomycin-resistant enterococci. An article of manufacture according tothe present invention can include primers and probes used to detectnucleic acids from vancomycin-resistant enterococci, together withsuitable packaging material. Representative primers and probes providedin a kit for detection of vancomycin-resistant Enterococcus can becomplementary to vanA, vanB, and/or vanB-2/3 nucleic acid molecules.Methods of designing primers and probes are disclosed herein, andrepresentative examples of primers and probes that amplify and hybridizeto vanA, vanB, and/or vanB-2/3 nucleic acid molecules are provided.

Articles of manufacture of the invention also can include one or morefluorescent moieties for labeling the probes or, alternatively, theprobes supplied with the kit can be labeled. For example, an article ofmanufacture of the invention may further include a donor fluorescentmoiety for labeling one of the vanA, vanB, and/or vanB-2/3 probes and acorresponding acceptor fluorescent moiety for labeling the other vanA,vanB, and/or vanB-2/3 probe, respectively. Examples of suitable FRETdonor fluorescent moieties and corresponding acceptor fluorescentmoieties are provided herein.

Articles of manufacture of the invention also can contain a packageinsert having instructions thereon for using pairs of vanA, vanB, and/orvanB-2/3 primers and vanA, vanB, and/or vanB-2/3 probes to detectvancomycin-resistant Enterococcus in a biological sample. Articles ofmanufacture additionally may include reagents for carrying out themethods disclosed herein (e.g., buffers, polymerase enzymes, co-factors,or agents to prevent contamination). Such reagents may be specific forone of the commercially available instruments described herein.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Sample Preparation

One hundred clinical isolates of Enterococcus (E.casseliflavus/flavescens [n=10], E. faecalis [n=34], E. faecium [n=43],E. avium [n=1], E. gallinarum [n=11], and E. raffinosus [n−−1]) weregrown on blood agar plates and evaluated for the presence of vanA, vanB,and vanB 2/3 genes using a rapid LIGHTCYCLER™ vancomycin-resistantenteroccoci assay. The LIGHTCYCLER™ vancomycin-resistant enteroccociassay differentiates the three van genes based on the temperature rangeover which hybridization probes melt ftom the target DNA (e.g., asdetermined by a melting curve). Results generated using the LIGHTCYCLER™vancomycin-resistant enteroccoci assay were compared to those obtainedusing a multiplex PCR-RFLP assay (Patel et al., 1997, J. Clin.Microbiol., 35:703) and agar dilution antimicrobial susceptibilitytesting following the current guidelines of the National Committee forClinical Laboratory Standards (NCCLS Document M7-A3, 1993. 57 of the 100isolates were negative for van genes and 43 were positive (11 vanA; 11vanB; 21 vanB-2/3) by the LIGHTCYCLER™ vancomycin-resistant enteroccociassay and the PCR-RFLP assay (11 vanA; 32 vanB). The MIC values for thevanA, vanB, and vanB-2/3 positive isolates were all resistant tovancomycin at ≧64 μg/ml. The MIC values for all of the other isolateswere susceptible to vancomycin at ≦8 μg/ml.

For each specimen culture, a 2.0 ml screw-capped tube was labeled, and300 μl of water was pipetted into each tube. Three to five colonies fromthe plate were pulled with a sterile loop and placed into water. Thetube was vortexed to mix. The tube was placed at 100° C. for 10 min andcentrifuged at 1000×g for 1 min.

Four hundred ninety seven anal swabs collected from hospitalizedpatients were evaluated for the presence of vanA, vanB and vanB-2/3genes by the LIGHTCYCLER™ vancomycin-resistant enterococci assay and byconventional culture methods. 482 of the 497 specimens were negative forthe van genes and 12 were positive (9 vanA; 0 vanB; 3 vanB-2/3) by theLIGHTCYCLER™ vancomycin-resistant enterococci assay and the conventionalculture screen using an 8 μg/ml Enterococcosel plate. The MIC values forthe vanA and vanB positive isolates were all resistant to vancomycin at≧64 μg/ml. The MIC values for all of the other isolates were susceptibleto vancomycin at ≦8 μg/ml. An additional 21 specimens (4 vanA; 5 vanB;12 vanB-2/3) were positive by the LIGHTCYCLER™ vancomycin-resistantenterococci assay but were negative by culture. Three of these patientspecimens were culture positive with a repeat swab collection. Six ofthe positive specimens were separate specimens from the same patients.Due to the large number of vanB genotypes missed by culture and pickedup with the LIGHTCYCLER™ vancomycin-resistant enterococci assay, theconcentration of vancomycin in the Enterococcesel plates was decreasedto 6 μg/ml, which is the usual laboratory standard (Manual of ClinicalMicrobiology, 7^(th) Ed., 1999).

For each patient specimen obtained from an anal or stool swab, a 2.0 mlscrew-capped tube was labeled with an accession label. 300 μl of waterwas pipetted into each tube. 1 vial of 0.1 mm zirconium beads (Daigger &Co., Inc.) was added to each tube. The swab was rinsed in the water andbeads. Any liquid remaining in the swab was expressed against the sideof the tube. The tubes were placed into the FastPrep apparatus andprocessed at a speed of 6.0 for 30 sec. Tubes were then centrifuged in abench top centrifuge at 20,800×g for 1 min.

For nucleic acid extraction from an anal or stool specimen, 100 μl STARbuffer ( ) was added to 100 μl of each vancomycin-resistant Enterococcalspecimen. Nucleic acid was then extracted using MagNA Pure ExtractionSystem (Roche applied Sciences), High Pure PCR Template Preparation kit(Roche Applied Sciences, or QIAamp DNA Stool Kit (Qiagen, Inc). Positiveand negative controls in STAR buffer (0.2 M citrate, 0.2 M EDTA, 0.5%ammonium lauryl sulfate, pH 5.0) were included with each nucleic acidextraction.

Example 2 Primers and Probes

Primers and probes directed toward vanA, vanB, and vanB2/3 were designed(Table 3). The vanA amplification product was 232 bp in length, the vanBamplification product was 167 bp in length, and the vanB-2/3amplification product was 166 bp in length. TABLE 3 Sequences of vanA,vanB, and vanB-2/3 primers and probes Primers vanA 5′-CGA GGA CGG ATACAG GA-3′ 1 5′-CTT ATC ACC CCT TTA ACG C-3′ 2 vanB/ 5′-GAA GAT ACC GTGGCT CA-3′ 5 vanB- 5′-GTA CGG AAG AAC TTA ACG CT-3′ 6 2/3 SEQ ID NO:Probes vanA 5′-CAA GAT AAC GGC CGC ATT GTA 3 CTG AAC GA-3′ 5′-GTC AATACT CTG CCC GGT TTC 4 AC-3′ vanB/ 5′-GAT CCA CTT CGC CGA CAA-3′ 7 vanB-5′-AAA TCA TCC TCG TTT CCC AT-3′ 8 2/3

vanA, and vanB/ vanB-2/3 primers were synthesized by the Mayo CoreFacility on a 0.2 nm scale, and were quantitated by UV absorption at 260nm and mixed together to make a solution containing 25 μM of eachprimer.

Probes were synthesized by TIB Molbiol LLC (Adelphia, N.J.), and weredissolved in TE′ (10 mM Tris (pH 8.0), 0.1 mM EDTA) to a finalconcentration of 20 μM (resuspended according to manufacturer'sinstructions). The concentration of oligonucleotides and dye wasdouble-checked by UV absorption using the following equations(Biochemica 1:5-8, 1999):$\lbrack{dye}\rbrack = {{\frac{A_{dye}}{E_{dye}}\lbrack{oligo}\rbrack} = \frac{A_{260} - \left( {A_{260} \times \frac{E_{260{({dye})}}}{E_{dye}}} \right)}{\frac{10^{6}}{{nmol}\text{/}A_{260}}}}$

Example 3 Detection Assays

For LIGHTCYCLER™ amplification to detect vancomycin-resistantenterococci in a stool sample, the following protocol was followed. TheLIGHTCYCLER™ vancomycin-resistant enterococci master mix (Table 4a) wasthawed, vortexed briefly, and centrifuged for 30 sec at 20,800×g. Thetime reagents were left at room temperature was minimized. TheLIGHTCYCLER™ carousel was loaded with one cuvette per sample, twocuvettes for positive controls and the appropriate number of cuvettesfor negative controls to total 5-10% of the total number of samples. 15μl of the LIGHTCYCLER™ vancomycin-resistant enterococci Master Mix wasadded to each cuvette. 5 μl of the extracted nucleic acid was added toeach LIGHTCYCLER™ cuvette.

The LIGHTCYCLER™ vancomycin-resistant enterococci FastStart Master Mix(Table 4b) using LIGHTCYCLER™ FastStart DNA Hybridization probe kit wasalso tested and results were comparable to the Master Mix having thecomponents shown in Table 4a. The addition of a recovery template in theFastStart Master Mix was used to prevent misinterpretation of falsenegative results caused by inhibition of amplification. The recoverytemplate used was a synthetic double-stranded DNA molecule with primerbinding sites identical to the vanB target sequence and a unique probebinding region that allowed differentiation of recovery template fromthe target specific amplicon. The recovery template probes were labeledwith a LC705 dye which was read in channel F3. TABLE 4a LIGHTCYCLER ™vancomycin-resistant enterococci Master Mix Ingredient Stock Mix μlWater 967 MgCl₂ 50 mM 2 mM 80 10X Buffer 10X 1X 200 Primers-vanA 25 μM0.5 μM 40 Primers-vanB 25 μM 0.7 μM 56 Platinum Taq 5 U/μl 0.03 U/μl 12dNTP plus 10 mM 0.2 mM 40 BSA 2% 0.025% 25 Probe-vanA-FL 20 μM 0.2 μM 20Probe-vanA-R640 20 μM 0.2 μM 20 Probe-vanB/vanB-2/3-FL 20 μM 0.2 μM 20Probe-vanB/vanB-2/3-R640 20 μM 0.2 μM 20 Total volume -> 1500

TABLE 4b LIGHTCYCLER ™ vancomycin-resistant enterococci FastStart MasterMix Ingredient Stock Mix μl Water 707.3 MgCl₂ 25 mM 2.5 mM 200.0 FAS 1Reaction Mix* 10X 1X 200.0 Recovery Template 10X 1X 200.0 Primers - vanA25 μM 0.5 μM 40.0 Primers - vanB 25 μM 0.7 μM 56.0 RT Probes - FL/Red 24μM 0.2 μM 16.7 Probe - vanA-FL 20 μM 0.2 μM 20.0 Probe - vanA-R640 20 μM0.2 μM 20.0 Probe - vanB/vanB-2/3-FL 20 μM 0.2 μM 20.0 Probe -vanB/vanB-2/3-R640 20 μM 0.2 μM 20.0 total volume -> 1500*FAS reaction mix contains 1 mM MgCl₂

The carousel containing the samples was centrifuged in the LIGHTCYCLER™carousel centrifuge. The carousel was placed in the LIGHTCYCLER™thermocycler and the LIGHTCYCLER™ vancomycin-resistant enterococciprogram using the Master Mix shown in Table 4a was run (Table 5a). Table5b shows the run profile used with the FastStart Master Mix. The cyclingsteps were complete in approximately one hour. After completion of thecycling, cuvettes were removed from the carousel with the cuvetteextractor. The carousel was decontaminated in 10% bleach for 10 min,rinsed well with de-ionized water, and dried. TABLE 5a PCR cyclingconditions for the LIGHTCYCLER ™ vancomycin-resistant enterococci assaywith the Table 4a Master Mix Program Temp Name/ Transition AnalysisAnalysis Temp Time Rate Signal mode mode Cycles (° C.) (sec) (° C./sec)Acquisition Initial None 1 95 120 20 None PCR Quant. 40 95 0 20 None 5512 20 Single 72 12 20 None Melt Melt 1 95 0 20 None Analysis 45 60 20None 80 0 0.2 Continuous Cool None 1 35 0 20 None

The gains were set at 1, 5, and 15 for channels F1, F2, and F3,respectively. TABLE 5b PCR cycling conditions for the LIGHTCYCLER ™vancomycin-resistant enterococci assay with FastStart Master Mix ProgramTemp Name/ Transition Analysis Analysis Temp Time Rate Signal mode modeCycles (° C.) (sec) (° C./sec) Acquisition Initial None 1 95 600 20 NonePCR Quant. 45 95 10 20 None 55 10 20 Single 72 12 20 None Melt Melt 1 950 20 None Analysis 59 20 20 None 45 20 0.2 None 80 0 0.2 Continuous CoolNone 1 40 30 20 NoneThe gains were set at 1, 5, and 30 for channels F1, F2, and F3,respectively.

The data was analyzed using the LIGHTCYCLER™ Software. A PCR meltinganalysis was used to differentiate vanA, vanB, and vanB-2/3 based on theTm of the FRET probes. The probes targeting the vanA gene melt at67±2.5° C., while the probes targeting the vanB and the vanB-2/3 genemelt at 60±2.0, and 56±2.0° C., respectfully.

A sample with a melting peak at the same location as the positivecontrol was interpreted as positive. Positive samples were reported aspositive for the presence of one or morevancomycin-resistant enterococcitarget sequences. A sample in which the melting curve was not abovebaseline was negative for the presence of vancomycin-resistantenterococci DNA. A negative result does not necessarily negate thepresence of the organism or active disease.

Example 4 Results, Assay Validation, and Quality Control

Control experiments were performed to determine if the primers andprobes described herein for detecting vancomycin-resistant enterococcicross-reacted with DNA from similar organisms or from organisms commonlyfound in the specimens. For the crossreactivity panels, the presence ofmicroorganism DNA was initially confirmed by amplification of 16S rRNAand electrophoretic separation of the amplification product (Johnson,1994, Methods for General and Molecular Bacteriology, American Societyfor Microbiology, Washington D.C.). Stool Specificity Panel ID# OrganismSource LC VRE SP1 Bacteroides fragilis ATCC25285 negative SP2Fusobacterium nucleatum ATCC25559 negative SP3 Clostridium perfringensATCC13124 negative SP4 Bacteroides distasonis ATCC8503 negative SP5Eubacterium lentum ATCC43055 negative SP6 Bacteroides thetaiotaomicronsATCC29741 negative SP7 Bacteroides vulgatus ATCC29327 negative SP8Echerichia vulneris Lab Isolate negative SP9 Klebsiella pneumoniaeATCC700603 negative SP10 Streptococcus viridans QC Strain negative SP11Escherichia hermanii Lab Isolate negative SP12 Actinomyces pyogenes LabIsolate negative SP13 Proteus mirabilis QC Strain negative SP14Pleisomonas shigelloides Lab Isolate negative SP15 Salmonella Group BCAP-D-1-69 negative SP16 Pseudomonas aeruginosa ATCC27853 negative SP17Escherichia coli ATCC25922 negative SP18 Aeromonas hydrophila CAP-D-1-82negative SP19 Staphylococcus aureus ATCC25923 negative SP20 Yersiniaenterocolitica Lab Isolate negative SP21 Staphylococcus epidermidisMK214 negative SP22 Shigella flexnerii Lab Isolate negative SP23Citrobacter freundii Lab Isolate negative SP24 Salmonella species LabIsolate negative SP31 Encephalitozoon intestinalis CDC:V297 negativeSP32 Escherichia coli O157:H7 ATCC35150 negative SP33 Shigelladysenteriae CDC 82-002-72 negative SP34 Shigella sonnei ATCC25931negative SP35 Escherichia coli O142:K86(B):H6 ATCC23985 negative SP36Escherichia coli O70:K:H42 ATCC23533 negative SP37 Escherichia coliO7:K1(L):NM ATCC23503 negative SP38 Enterobacter cloacae ATCC13047negative

Panel of Isolates from Stool that are Vancomycin Resistant Panel ofIsolates from Stool that are Vancomycin Resistant ID# Organism Source LCVRF SP25 Streptococcus bovis CAP-D-16-83 negative SP26 Pediococcusspecies Lab Isolate negative SP27 Lactobacillus species QC Strainnegative SP28 Leuconostoc species Lab Isolate negative SP29Streptococcus bovis Lab Isolate negative SP30 Lactobacillus species LabIsolate negative SP39 Streptococcus bovis Lab Isolate negative SP40Streptococcus bovis Lab Isolate negative SP41 Leuconostoc species LabIsolate negative SP42 Pediococcus species Lab Isolate negative SP43Leuconostoc species Lab Isolate negative SP44 Lactobacillus species LabIsolate negative SP45 Lactobacillus species Lab Isolate negative SP46Leuconostoc species Lab Isolate negative SP47 Streptococcus bovis LabIsolate negative SP48 Streptococcus bovis Lab Isolate negative

Enterococcus Specificity Panel RFLP ID# Organism Source ResultLIGHTCYCLER ™ E1 Enterococcus Lab Isolate vanC-1 negative gallinarum E2Enterococcus Lab Isolate vanC-2/3 negative casseliflavus E3 EnterococcusLab Isolate negative negative faecalis E4 Enterococcus Lab IsolatevanC-1 negative gallinarum E5 Enterococcus Lab Isolate vanA and negativegallinarum C-1 E6 Enterococcus Lab Isolate negative negative faecium E7Enterococcus Lab Isolate vanC-1 negative gallinarum E8 Enterococcus LabIsolate vanC-1 negative gallinarum E9 Enrerococcus Lab Isolate negativenegative rhaffinosus E10 Enterococcus Lab Isolate vanC-2/3 negativecasseliflavus E11 Enterococcus Lab Isolate vanC-1 negative gallinarumE12 Enterococcus Lab Isolate negative negative faecalis E13 EnterococcusLab Isolate negative negative casseliflavus E14 Enterococcus Lab Isolatenegative negative faecium E15 Enrerococcus Lab Isolate negative negativefaecalis E16 Enterococcus Lab Isolate vanC-1 negative faecium E17Enterococcus Lab Isolate vanC-1 negative gallinarum E18 Enterococcus LabIsolate vanC-2/3 negative casseliflavus E19 Enterococcus Lab IsolatevanC-2/3 negative casseliflavus

The vanA, vanB, or vanB-2/3 primers and probes described herein did notcross-react with any of the above-indicated Enterococcus spp. or stoolisolates tested.

In addition, control experiments were performed to determine ifLIGHTCYCLER™ amplification from clinical samples produced a singleamplification product. Amplification products were analyzed by 2%agarose gel electrophoresis. In positive clinical specimens,amplification using the LIGHTCYCLER™ protocol generated a single band atthe expected size.

Additional control experiments were performed using dilutions ofpositive control plasmid to determine the sensitivity of theLIGHTCYCLER™ assay. Plasmid dilutions ranged from 0.2/μl up to2.0×10⁵/μl. Reactions were perfonned as described above. Data wasplotted as the level of fluorescence detected relative to the cyclenumber for each dilution value. The slope of the standard curve was−3.236 with an r value=−1.00. Using the formulas ExponentialAmplification=10^((−1/slope)), and Efficiency=(10^((−1/slope)))−1, theefficiency of the reaction was determined to be 1.037152. Thesensitivity of the LIGHTCYCLER™ reaction was less than 50 copies oftarget per 5 μl of sample with the PCR mix.

Further control experiments were performed to determine the sensitivityand specificity of the LIGHTCYCLER™ assay compared to a PCR-RFLP(restriction fragment length polymorphism) method. 60 clinical isolatesof enterococci previously examined for the presence of avancomycin-resistant gene by PCR-RFLP were tested using the LIGHTCYCLER™assay described herein. The isolates were grown on blood agar plates at37° C. overnight. Cells from enterococci colonies were lysed bysuspending the colony in 500 μl sterile water and boiling the sample at100° C. for 10 minutes. The tube then was centrifuged for 1 minute at20,800×g, and 5 μl of the supernatant was analyzed by the LIGHTCYCLER™assay described herein.

The LIGHTCYCLER™ assay described herein correlated 100% with the resultsfrom the PCR-RFLP assay (27 samples were positive forvancomycin-resistant enterococci; 33 samples were negative forvancomycin-resistant enterococci). Note that vanC genotypes are detectedwith the PCR-RFLP assay, although the current LIGHTCYCLER™ assay doesnot detect genotypes that are negative for the vancomycin-resistancephenotype. PCR- # RFLP LIGHTCYCLER ™ Organism 1 vanA vanA E. faecium 2vanA vanA E. faecium 4 vanA vanA E. faecium 5 vanB vanB E. faecium 6negative negative E. faecalis 7 negative negative E. faecalis 9 negativenegative E. faecalis 10 vanB vanB-2/3 E. faecium 11 vanA vanA E. faecium13 vanA vanA E. faecium 15 vanB vanB-2/3 E. faecium 16 vanB vanB E.faecium 17 vanB vanB E. faecium 19 negative negative E. faecalis 20negative negative E. faecalis 21 vanC-2/3 negative E. casseliflavus 23negative negative E. avium 25 negative negative E. faecalis 28 vanB vanBE. faecium 29 negative negative E. faecalis 30 negative negative E.faecalis 31 vanB vanB E. faecium 32 vanC-2/3 negative E. casseliflavus33 vanB vanB-2/3 E. faecium 34 vanB vanB-2/3 E. faecium 35 vanB vanB E.faecalis 36 negative negative E. faecium 38 vanB vanB-2/3 E. faecium 39negative negative E. faecalis 40 negative negative E. faecalis 41 vanCnegative E. faecalis 42 vanC-1 negative E. gallinarum 44 vanC-2/3negative E. casseliflavus 45 vanB vanB-2/3 E. faecalis 46 vanB vanB-2/3E. faecium 47 vanB vanB-2/3 E. faecium 48 negative negative E. faecalis49 negative negative E. faecalis 50 negative negative E. faecalis 51vanB vanB E. faecium 54 vanB vanB-2/3 E. faecium 55 vanB vanB-2/3 E.faecium 56 vanB vanB E. faecalis 57 negative negative E. faecalis 58negative negative E. faecium 59 negative negative E. faecalis 60negative negative E. faecalis 61 vanA vanA E. faecium 62 vanB-v vanB-2/3E. faecium 63 vanA vanA E. faecium 64 negative negative E. faecalis 65negative negative E. casseliflavus 66 vanC-1 negative E. gallinarum 67vanB vanB-2/3 E. faecium 68 vanC-1 negative E. gallinarum 69 negativenegative E. faecalis 70 negative negative E. faecalis 71 negativenegative E. faecalis 72 vanC-2/3 negative E. casseliflavus 74 negativenegative E. faecium

An additional 56 specimens obtained from anal swabs were analyzed usingthe LIGHTCYCLER™ assay and were compared to a culture method using a VREscreen media. The anal swabs were prepared for amplification asdescribed above. The LIGHTCYCLER™ assay detected more positive specimensthan did culture, and is therefore more sensitive than culture. A highrate of false negative results from rectal swab culture has beenpreviously confirmed in the literature (Clin. Infect. Dis., 2002,34:167-172). Culture (Gold Standard) VRE Positive VRE Negative TOTALLIGHTCYCLER ™ VRE Positive 7 5 12 VRE Assay VRE Negative 0 44 44 TOTAL 749 56

# Culture Results^(a) Susceptibility Results^(b) LIGHTCYCLER ™ Results52 Enterococcus Amp, Pen, Gent = S; Van = I VanA 129 NG VanB 235Enterococcus Amp, Van, Pen, Gent = R VanA 244 Enterococcus Amp, Van, Pen= R; Gent = S VanA 279 Enterococcus Amp, Pen = R; Van, Gent = S VanB-2/3280 NG Negative 281 NG Negative 282 NG Negative 286 Enterococcus Amp,Van, Pen, Gent = S VanB-2/3 (weak) 287 NG Negative 296 Enterococcus Amp,Van, Pen, Gent = S Negative 298 NG Negative 299 NG Negative 300 NGNegative 301 NG Negative 302 NG Negative 303 NG Negative 304Enterococcus Amp, Pen, Gent = S; Van = I Negative 305 NG Negative 306 NGNegative 307 NG VanB-2/3 308 NG Negative 309 NG Negative 310 NGFollow-up specimen positive VanB-2/3 311 NG Negative 319 NG Negative 338NG Negative 348 NG Negative 419 Enterococcus Amp, Van, Pen = R; Gent = IVanA 420 NG Negative 421 NG Negative 422 NG Negative 423 NG Negative 424NG Negative 425 Enterococcus Amp, Pen, Gent = S; Van = I Negative 426 NGNegative 448 NG Negative 449 NG VanB-2/3 (weak) 450 NG Negative 451 NGNegative 452 NG Negative 453 NG VanB 454 NG Negative 455 NG Negative 469NG Negative 470 NG Negative 471 NG Negative 472 NG Negative 473 NGVanB-2/3 474 NG Negative 475 NG Negative 476 NG Negative 525 NG Negative526 NG Negative 527 NG VanB-2/3 528 NG Negative^(a)NG, No growth.^(b)Amp, Ampicillan; Pen, Pennicillin; Gent, Gentamycin; Van,Vancomycin.^(b)R, Resistant; S, Susceptible; I, Intermediate.

Control experiments also were performed to determine the precision(e.g., within-run, within-day, and between-day precision) of theLIGHTCYCLER™ assay. Within-run precision of the LIGHTCYCLER™ assay wasevaluated by assaying 5 μl of a positive control dilution 20 timeswithin the same amplification experiment. Within-day precision of theLIGHTCYCLER™ assay was evaluated by assaying 5 μl of a positive controldilution 20 times during a single day. Between-day precision of theLIGHTCYCLER™ assay was evaluated by assaying 5 μl of positive controldilution 20 times over a three-day period.

The average number of cycles at which FRET was detected in thewithin-run assays was 30.82±0.293; the average number of cycles at whichFRET was detected in the within-day assays was 30.61±0.190; and theaverage number of cycles at which FRET was detected in the between-dayprecision was 30.61±0.190 (day1), 30.17±0.154 (day2), and 29.41±0.143(day3). The precision of the average crossing point measurement and thestandard deviation of the 20 points was excellent.

Control experiments were performed to determine if the LIGHTCYCLER™assay produces the same results using 2, 5 or 10 μl of the nucleic acidsample extracted from a patient's sample. Mixes were prepared fordifferent target volumes essentially as described above, and a set of 2positive samples (vanA and vanB-2/3) was tested at each volume. Similarresults were obtained from patient specimens when 5 or 10 μl of samplewas used in the assay. The cross points were within one cycle. Adifference of 2-3 cycles was observed when 2 μl of sample was used.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An article of manufacture, comprising: a pair of vanA primers; a pairof vanA probes; and a donor fluorescent moiety and a correspondingfluorescent moiety.
 2. The article of manufacture of claim 1, whereinsaid pair of vanA primers comprises a first vanA primer and a secondvanA primer, wherein said first vanA primer comprises the sequence5′-CGA GGA CGG ATA CAG GA-3′ (SEQ ID NO:1), and wherein said second vanAprimer comprises the sequence 5′-CTT ATC ACC CCT TTA ACG C-3′ (SEQ IDNO:2).
 3. The article of manufacture of claim 1, wherein said pair ofvanA probes comprises a first vanA probe and a second vanA probe,wherein said first vanA probe comprises the sequence 5′-CAA GAT AAC GGCCGC ATT GTA CTG AAC GA-3′ (SEQ ID NO:3), and wherein said second vanAprobe comprises the sequence 5′-GTC AAT ACT CTG CCC GGT TTC AC-3′ (SEQID NO:4).
 4. The article of manufacture of claim 1, wherein said pair ofvanA probes comprises a first vanA probe labeled with said donorfluorescent moiety and a second vanA probe labeled with saidcorresponding acceptor fluorescent moiety.
 5. The article of manufactureof claim 1, further comprising a package label or package insert havinginstructions thereon for using said pair of vanA primers and said pairof vanA probes to detect the presence or absence of one or morevancomycin-resistant enterococci in a biological sample.
 6. An articleof manufacture, comprising: a pair of vanlb primers; a pair of vanBprobes; and a donor fluorescent moiety and a corresponding fluorescentmoiety.
 7. The article of manufacture of claim 6, wherein said pair ofvanB primers comprises a first vanB primer and a second vanB primer,wherein said first vanB primer comprises the sequence 5′-GAA GAT ACC GTGGCT CA-3′ (SEQ ID NO:5), and wherein said second vanB primer comprisesthe sequence 5′-GTA CGG AAG AAC TTA ACG CT-3′ (SEQ ID NO:6).
 8. Thearticle of manufacture of claim 6, wherein said pair of vanB probescomprises a first vanB probe and a second vanB probe, wherein said firstvanB probe comprises the sequence 5′-GAT CCA CTT CGC CGA CAA-3′ (SEQ IDNO:7), and wherein said second vanB probe comprises the sequence 5′-AAATCA TCC TCG TTT CCC AT-3′ (SEQ ID NO:8).
 9. The article of manufactureof claim 6, wherein said pair of vanB probes comprises a first vanBprobe labeled with said donor fluorescent moiety and a second vanB probelabeled with said corresponding acceptor fluorescent moiety.
 10. Thearticle of manufacture of claim 6, further comprising a package label orpackage insert having instructions thereon for using said pair of vanBprimers and said pair of vanB probes to detect the presence or absenceof vancomycin-resistant enterococci in a biological sample.